Cells within many tissues and organs are continually replenished throughout our lives. This is accomplished by stem cells, which balance self-renewal with more differentiated cell types. The mechanisms that control this balance are not clear. In tissues maintained by stem cells, the differentiating daughters undergo mitotic expansion before generating tissue-specific cell types. The mechanisms that control transit amplification are also not clear. Both of these phenomena are featured in Drosophila spermatogenesis, where stem cell daughters choose between self-renewal and differentiation, and where transit amplifying gonial cells switch to spermatocyte development. Gonial cells divide four times; the counting and effector mechanisms regulating this are unknown. Aim 1 tests the hypothesis that germ cells count intrinsically, and then collaborate with surrounding somatic cells to coordinate the spermatocyte transition. We discovered that a somatic cell signal promotes differentiation of stem cell daughters, while other labs discovered a signal that promotes self-renewal. Aim 2 tests the hypothesis that self-renewal and differentiation are indeed balanced by these competing signals, and then investigate how they compete. To generate and begin to test biologically based hypotheses for other candidate factors and signaling pathways that legislate between renewal and differentiation we have conducted transcript-profiling analyses of self-renewing cells or their differentiating daughters. Aim 3 proposes to complete these studies and conduct functional tests on selected candidates.